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Environmental and Experimental Botany 59 (2007) 179–187

Growth and protein profile changes in Lepidium sativumL. plantlets exposed to cadmium

Elisabetta Gianazza a, Robin Wait b, Andrea Sozzi a, Simona Regondi a,Dolores Saco d, Massimo Labra c, Elisabetta Agradi a,∗

a Universita degli Studi di Milano, Dipartimento di Scienze Farmacologiche,Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Via G. Balzaretti, 9, 20133 Milano, Italy

b Kennedy Institute of Rheumatology Division, Imperial College London, 1, Aspenlea Road, Hammersmith, London W6 8LH, United Kingdomc Universita degli Studi di Milano-Bicocca, Dipartimento di Scienze dell’Ambiente e del Territorio, P.zza della Scienza, 1, 20126 Milano, Italy

d Universidad Complutense de Madrid, Departamento de Biologia Vegetal II, Facultad de Farmacia, 28040 Madrid, Spain

Received 25 May 2005; received in revised form 4 October 2005; accepted 20 December 2005

bstract

Plant metabolic response to heavy metal stress is still to be clarified. The present investigation was undertaken to examine the influence ofifferent concentrations of cadmium on the Lepidium sativum L. plantlets. Exposure of seedlings of L. sativum L. to increasing concentrationsf cadmium results in the growth inhibition and in the accumulation of proteins in the 10–25 kDa range in cotyledons and hypocotyls of thelantlets. Most of these proteins are also found in extracts of L. sativum seeds. Analysis by ESI-MS after two-dimensional electrophoresis showedhat these proteins exhibit sequences similar to those of storage proteins from various Cruciferae species. The response to metal exposure duringermination and initial plantlet elongation thus involves inhibition of both storage protein catabolism and plant protein anabolism. In addition,wo of the proteins present in higher amounts in plantlets exposed to cadmium heat-shock, in agreement with literature data, and jasmonate-like

nducible protein are related to cellular stress and another two (LEAs or late embryogenesis abundant) are involved in embryogenesis. Changesn protein expression can be detected by two-dimensional electrophoresis after exposure to heavy metal concentrations lower than those at which

orphometric changes become evident. Proteomics of germinating L. sativum thus constitutes a very sensitive tool for evaluating environmentalollution.

2006 Elsevier B.V. All rights reserved.

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eywords: L. sativum; Cadmium; Biomarker; Proteomics; Growth inhibition

. Introduction

Bioassay reflects toxicological damage at biochemical andhysiological levels; furthermore, it can help to define new sen-itive and specific biomarkers.

Plants are good bioindicators because they play a significantole in food chain transfer and in defining environmental health.hey are easy to grow and adaptable to environmental stress andlso reflect toxicant damage in other organisms, such as animalsMinissi and Lombi, 1997). It has been shown that they are very

ensitive to some specific stressors (Wang and Freemark, 1995).

The effects of heavy metals on plant and plant defense sys-ems have been investigated intensively at genomic level and

∗ Corresponding author. Tel.: +39 0250318294; fax: +39 0250318284.E-mail address: elisabetta.agradi@unimi.it (E. Agradi).

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098-8472/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2005.12.005

eviewed by various authors (Prasad, 1995; Das et al., 1997;anita di Toppi and Gabrielli, 1999; Labra et al., 2003, 2004).hromosome aberration assays, mutation assays, cytogenetic

ests and specific locus mutation assays have been performedConstantin and Nilan, 1982; Tardiff et al., 1994; Marcon et al.,999) with different plant species.

In recent years, new techniques such as transcriptomicsased DNA-microarrays (Lange and Ghassemian, 2005) androteomics have been developed to evaluate environmentallynduced protein changes in living organisms. Proteomics is thetudy of the protein complement of the genome and has beenpplied mainly to drug discovery and protein interaction in bio-hemical pathways (Jain, 2001; Walgren and Thompson, 2004),

lthough there is increasing interest in its use in diagnosis.he basic principle is that every condition produces a uniqueet of proteins in the exposed organisms. Proteins are the pri-ary effector molecules of all living systems, and therefore

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80 E. Gianazza et al. / Environmental an

irtually any adaptive response to environmental, physiologi-al or pathological conditions will be reflected by alterationsn protein activity, location and concentration (Shepard et al.,000; Bradley et al., 2002). Moreover, plant responses to heavyetal toxicity include the expression of stress proteins and phy-

ochelatins (Cobbett, 2000).The aim of this study is to construct a protein map related

o Lepidium sativum L. plant, showing the protein expressionariation in response to specific heavy metals. As indicated byECD, L. sativum exposed to heavy metals during germination,nder standardized conditions, is a useful model of environ-ental stress (OECD, 1984). In fact, toxicological insult causes

rowth inhibition rather than tissue necrosis and plant death,ven over a wide range of metal concentration.

In this paper, we report changes in the proteome of L. sativumn response to cadmium. Cadmium was selected since it is aidely occurring toxic heavy metal, classified as a human car-

inogen by the International Agency for Research on Cancern 1993 (IARC, 1993; Beyersmann and Hechtenberg, 1997;

aalkes, 2000; Waisberg et al., 2003). Plants also easily absorbadmium.

Data obtained from our work will contribute to a better under-tanding of the mechanisms involved in metal toxicity and mayead to the discovery of new biomarkers to be used in monitoringrotocols.

. Materials and methods

.1. Culture conditions and growth evaluation

L. sativum L. seeds were obtained from F.lli IngegnoliMilano, Italy); a sample specimen is deposited at Departmentf Biology, University of Milan, Italy. The L. sativum seedsere germinated in disposable Petri dishes, 100 mm in diam-

ter, on ashless Whatmann filter paper moistened with 5 mLf either double-distilled (dd) water (control) or cadmium testolution. The test was performed on plantlets exposed to increas-ng concentrations (2, 5, 10, 20, 50, 100 and 200 mg L−1) ofadmium chloride (Sigma, St. Louis, MO). Concentration inerms of weight-by-volume is defined in the reference protocolf the Intercalibration Action sponsored by the Italian Nationalesearch Council (CNR, Rome); in molar terms, 200 mg L−1

orresponds to 1.1 × 10−3 of cadmium. Tests were run in qua-ruplicate, on 10 seeds per dish. Petri dishes were kept in theark, at 25 ◦C, for 72 h.

The length of the whole plantlet and of root, hypocotyl andpicotyl was measured with a ruler (against a black background);verage values and standard deviation (S.D.) were evaluated forach replicate (dish). Dry weight was evaluated after drying thepecimens (40 seeds) for 72 h at 76 ◦C.

.2. Protein extraction

All extractions were normalized on the basis of fresh weighto buffer ratio. Whole and dissected (seed teguments, root,ypocotyl, cotyledons) plantlets were extracted with sampleuffer under reducing conditions (2%, v/v, 2-mercaptoethanol)

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erimental Botany 59 (2007) 179–187

or SDS-PAGE according to Laemmli (1970), at a buffer-to-issue ratio of 9:1. After grinding in a homogenizer (Ultraturrax25, Janke & Kunkel-IKA, Taufen, D) the suspension was soni-ated three times for 30 s and centrifuged for 5 min at 15,000 rpmEppendorf centrifuge, Brinkmann, Westbury, NY). Seeds wererushed in a mortar; a 1:18 sample:buffer suspension was thentirred for 30 min at room temperature before centrifugation asbove. After boiling for 5 min either the collected supernatantsere stored at −80 ◦C till used or proteins were precipitatedith cold acetone (9 volumes at −20 ◦C).

.3. 1-D electrophoresis

Aliquots of 50 �L of the plantlet extracts were loaded on–20%T PAA gradients for SDS-PAGE and run in the discon-inuous buffer system of Laemmli (1970). Proteins were stainedith a 0.3% (w/w) solution of Coomassie Brilliant Blue R-250 in0% ethanol:10% acetic acid:60% water (v/v/v). The resultingatterns were scanned with a CCD camera (Sony). The inten-ity of the various bands/band groups was measured with NIHmage (release 1.62) software (W. Rasband, NIH, Bethesda,SA).

.4. 2-D electrophoresis

Acetone pellets were dissolved in 8 M urea, 2% 2-ercaptoethanol. For 1-D fractionation by charge, the protein

olutions were loaded at the cathode edge on immobilized pHradient (IPG) strips (Bjellqvist et al., 1982; Gianazza, 2002)overing the pH range 4–10 non-linearly (Gianazza et al., 1985).ocusing was for a total of 15,000 V h−1 at 15 ◦C, in a MultiphorI horizontal cell (Amersham Biotech, Uppsala, USA). Afterquilibration for 15 min in 3% SDS—2% 2-mercaptoethanol,he IPG strips were embedded with 0.7% agarose on 7.5–17.5%TAA gradient slabs. For 2-D fractionation by size, SDS-PAGEas run in a vertical cell (Protean II, BioRad, Hercules, CA),

t 50 mA per slab. Proteins were stained with Coomassie asbove.

The spots whose abundance was found to vary extensivelymore than 200%) between control and test extract were excisedor identification by mass spectrometry (MS).

.5. Mass spectrometry

In-gel digestion with trypsin was performed according to pub-ished methods (Jeno et al., 1995; Wilm et al., 1996; Shevchenkot al., 1996) modified for use with a robotic digestion systemGenomic Solutions, Huntington, UK). Cysteine residues wereeduced with DTT and derivatized by treatment with iodoac-tamide. The gel pieces were then dehydrated with acetoni-rile and dried at 60 ◦C prior to addition of modified trypsinPromega, Madison, WI; 10 �L at 6.5 ng �L−1 in 25 mM ammo-ium hydrogen carbonate). After incubation at 37 ◦C for 8 h,

he products were sequentially extracted with 25 mM ammo-ium hydrogen carbonate, 5% formic acid and acetonitrile.yophilized extracts were re-dissolved in 0.1% formic acid prior

o MALDI and ESI-MS/MS analysis.

d Experimental Botany 59 (2007) 179–187 181

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E. Gianazza et al. / Environmental an

MALDI mass spectra were obtained with a TofSpec 2Epectrometer (Micromass, Manchester, UK) using �-cyano--hydroxycinnamic acid matrix as described (Wait et al.,001).

Tandem electrospray mass spectra were recorded using a-Tof spectrometer (Micromass, Manchester, UK) interfaced

o a CapLC Capillary Chromatograph (Micromass). Samplesere injected onto a 300 �m × 15 mm Pepmap C18 column

LC Packings, Amsterdam, NL), and eluted with an acetoni-rile/0.1% formic acid gradient. The capillary voltage was seto 3500 V, and data dependant MS/MS acquisitions were per-ormed on precursors with charge states of 2, 3 or 4 over aurvey mass range 540–1000. The collision gas was argon, andhe collision voltage was varied between 18 and 45 V depend-ng on the charge-state and mass of the precursor. Initial proteindentifications were made by correlation of uninterpreted tan-em mass spectra to entries in SwissProt and TREMBL usingroteinLynx Global Server (v. 1.0 and 1.1, Micromass). Oneissed cleavage per peptide was allowed, and the fragment ion

olerance was set to 100 ppm. Carbamidomethylation of cys-eine was assumed, but other potential modifications were notonsidered in the first pass search. All matches were reviewedanually, and in cases were the score reported by ProteinLynxlobal Server was less than 100, additional searches were per-

ormed against the NCBI non-redundant database using MAS-OT (Perkins et al., 1999), which utilizes a robust probabilistic

coring algorithm. When these approaches failed, amino acidequences were deduced manually from the charge state de-ncrypted spectra, and searched against the NCBI’s n-r databasesing BLAST (Altschul et al., 1997) and FASTS (Mackey et al.,002).

. Results

.1. Growth inhibition

In this study, L. sativum seeds were exposed to increasingoncentrations of cadmium, ranging from 2 to 200 mg L−1 andfter 72 h the root, hypocotyl and epicotyl length were measured.

Fig. 1 shows the effect of different cadmium concentrationsn the elongation of germinating L. sativum plantlets. Growthnhibition appears to be concentration-dependent. Inhibition of0% is observed for concentrations of 50 mg L−1 of cadmiumhloride (0.3 mM cadmium).

Fig. 2 shows the varying effects of cadmium toxicity onhe different anatomical parts of L. sativum plantlets. In panel, length (mm) is compared for roots, hypocotyls, epicotyls

nd whole plantlets. At the highest metal concentration, theength of the roots is reduced to 1/10 compared to control, thatf hypocotyls to 1/5, whereas epicotyls undergo insignificanthanges. Whole plantlet length is reduced to 1/4 of control. Panelreports the dry weight of the different anatomical parts. Differ-

nces between samples are not very large. A positive correlation

ith cadmium exposure is observed for epicotyls.Data obtained from our experiments show that L. sativum is

ensitive to cadmium. In addition, our data show that the selectedadmium concentrations induced a clear physiological modifi-

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ig. 1. Percent inhibition of L. sativum plantlet elongation (whole plantlets), asfunction of exposure to cadmium (logarithmic x-axis).

ation in L. sativum plants and these experimental conditionsepresented a suitable treatment for proteomics analysis.

.2. Changes in proteomic pattern

Whole and dissected (seed teguments, root, hypocotyl,otyledons) plantlets were used for protein extractions. Fig. 3hows the protein pattern of whole plantlet extracts from L.ativum grown under control conditions (lane A) and afterxposure to 200 mg L−1 cadmium chloride (lane B). Therere obvious differences in protein abundance, the most sig-ificant of which is an increase in levels of proteins in the0–25 kDa range after cadmium treatment. For densitometricvaluation protein bands were arranged into six groups (I–VI;ig. 3, lane C).

Band group IV (which includes the main storage proteinsdentified by mass spectrometry) increases approximately lin-arly with concentration after exposure to cadmium (Fig. 4).o consistent change is observed below 100 mg L−1 of cad-ium. Above 100 mg L−1 the abundance of band group IV

ncreases sharply. For these reasons, 2-D electrophoresis anal-sis was performed on plants treated at the 200 mg L−1 ofdCl2.

.3. Proteomic data from 2-D electrophoresis

Fig. 5 compares the 2-DE patterns obtained from controlpanel A) and cadmium-exposed (panel B) L. sativum plantlets.ll proteins present at higher concentrations in the exposed

pecimen are resolved into spot rows. Spots selected for in-el digestion and characterization by MALDI and electrosprayass spectrometry are indicated in Fig. 6. Identification byALDI mass fingerprinting was unsuccessful in all cases, pre-

umably because of the absence of the target sequences fromwissProt/TREMBL, but the determination of partial aminocid sequences by ESI-MS enabled retrieval of hits corre-ponding to related proteins from other plant species. The

182 E. Gianazza et al. / Environmental and Experimental Botany 59 (2007) 179–187

Fig. 2. Changes in length (panel A) and dry weight (panel B) of L. sativumplantlets exposed to increasing concentrations of cadmium (logarithmic x-axis).It

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Fig. 3. SDS-PAGE on a 4–20%T PAA gradient of whole L. sativum plantletextracts: (A) control growth conditions (72 h at 25 ◦C in the dark, dd water);(B) germinating seeds exposed to 200 g L−1 CdCl2. Fifty microliters of a 1:9extract per lane. Mr scale on the left; cropped images, as no protein componentwf

sspectgments, in either control or cadmium-exposed samples (Fig. 7,lane B).

n (A), squares refer to roots, diamonds to hypocotyl, circles to epicotyls andriangles to whole plantlets.

ata in Table 1 show sequence similarities between cadmium-ependent proteins from L. sativum plantlets and storage pro-eins from various Cruciferae species. No matching sequenceould be found for 7 out of 42 spots (Fig. 3). Consistentith this observation, several spots in heavy metal exposed. sativum migrate identically to the major spots of a seedxtract when analyzed by 2-DE (Fig. 5, panel C). However,he spots of the lowest Mr rows (27–43, Fig. 6) are absentrom seed extracts. The most likely explanation is that thesepots correspond to large, reproducible fragments of the storageroteins.

.4. Plantlet dissection data

To investigate the anatomical localization of storage proteins,ontrol and cadmium-exposed specimens were carefully dis-

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ith Mr > 100 kDa was observed in L. sativum samples. In (C), band groupingor quantitative procedures.

ected. In control plants significant levels of proteins with theame molecular mass as those in seeds (Fig. 7, lane A) wereresent only in cotyledons (lane C). In contrast, in pollutant-xposed L. sativum, storage proteins were found not only inotyledons (Fig. 7, lane E), where their concentration was higherhan in controls, but also in hypocotyls (Fig. 7, lane F). Afterermination, virtually no protein was detected in seed tegu-

ig. 4. Percent change of the (absolute) intensity of band group IV against con-rol, as a function of exposure to cadmium (logarithmic x-axis). Densitometricata on SDS-PAGE patterns of extracts from whole L. sativum plantlets exposedo increasing concentrations of cadmium.

E. Gianazza et al. / Environmental and Experimental Botany 59 (2007) 179–187 183

Fig. 5. 2-DE on non-linear 4–10 IPG followed by SDS-PAGE on 7.5–17.5%TPAA. (A) Extract of whole L. sativum plantlets grown under control conditions(acetone pellet from 1 mL of a 1:9 extract); (B) extract of whole L. sativumpe2

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lantlets exposed to 200 mg L−1 CdCl2 (acetone pellet from 1 mL of a 1:9xtract); (C) extract of L. sativum seeds treated whit Cd (acetone pellet from00 �L of a 1:18 extract).

. Discussion

.1. Proteomics with incomplete genomic databases

Genomic and proteomic investigation of green plants is lessdvanced than for animals or prokaryotes. Currently, the onlyigher plants whose genomes have been completely sequenced

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AA of the acetone pellet from 1.5 mL of a 1:9 extract of whole L. sativumlantlets exposed to 200 mg L−1 CdCl2. Circled spots were excised for MSnalysis (results in Table 1).

re Arabidopsis thaliana and Oryza sativa. Very few proteinsrom Lepidium species have been sequenced; at present Swis-Prot and TREMBL have only nine entries, and most of thesere partial sequences rather than full-length gene products. Thisaucity of sequence data has implications for proteomic investi-ation, because mass spectrometric protein identification reliesn comparison of experimental data with the computationallyredicted properties of database sequences.

Identification by MALDI peptide mass fingerprinting, forxample, requires the target protein, or a close relative, to beresent in the database, and cross-species identification is possi-le only for highly conserved proteins with substantial regionsf local identity. Unsurprisingly, therefore, no statistically sig-ificant hits were obtained by searching the MALDI spectra of2 excised spots against the NCBI n-r database (data not shown).

The use of amino acid sequence data rather than peptideasses to interrogate protein databases enables the use of error

olerant search strategies which can retrieve related but non-dentical proteins from other organisms, even when the targetrotein is not present in a database. The superiority of tandemS compared to MALDI for proteomic analysis of plant speciesith incompletely sequenced genomes was demonstrated by a

ecent study of endoplasmic reticulum from developing seeds oficinus communis (Maltman et al., 2002).

In the present study, ESI-MS/MS enabled matching of 32 outf 42 excised spots to homologous proteins from other plantpecies. These 32 gel features represent the products of only 13istinct genes. This is consistent with the high incidence of spotows in the 2-D map (Fig. 6) corresponding to a series of pro-eins, differing by charge, attributable either to small differencesn primary structure (e.g. point mutations), to post translational

odification, or to deamidation (Sarioglu et al., 2000). Thisxtensive charge microheterogeneity and the significant hetero-eneity in apparent mass are common findings in plant storage

roteins, in agreement with actual spot identifications. Althoughhe need for close packing under conditions of limited hydrationn seeds imposes some structural constraints, the selective pres-ure on these proteins appears much less severe than on other

184 E. Gianazza et al. / Environmental and Experimental Botany 59 (2007) 179–187

Fig. 7. SDS-PAGE on a 4–20%T PAA gradient of the extracts from different L. sativum samples. (A) Dry seeds (15 �L of a 1:18 extract); (B) seed remnants afterg tern fc (60 �L( dCl2

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ermination (1 mL 1:4 extract, concentrated by acetone precipitation; same patontrol plantlets (30 �L of a 1:9 extract); (D) hypocotyls from control plantlets30 �L of a 1:9 extract); (F) hypocotyls from plantlets exposed to 200 mg L−1 C

roteins. Since many of their genes occur in multiple copies tonable rapid protein deposition in the seed, sequence divergencean easily accumulate.

All except 1 of the retrieved sequences corresponded toroteins from Brassicaceae, including 22 from A. thaliana (7ene products), 11 from Brassica napus (4 genes), and 1 fromrassica campestris; the other protein was similar to 1 fromossipium hirsutum, a Malvaceae; all identified spots were thusomologues of proteins from Rosidae.

While storage proteins (subunits of 11S/12S globulins) con-tituted the majority of identified species, two (low molec-lar weight heat-shock and jasmonate-like inducible protein)ere clearly connected to cellular stress and two (LEA, or late

mbryogenesis abundant) were involved in embryogenesis.

.2. Heavy metal toxicity in plants

The presence of substantial amounts of unmodified or min-mally modified storage proteins in heavy metal exposed L.ativum, in parallel with a reduction in plantlet elongation, sug-ests that inhibition of storage protein catabolism and of plantletrotein anabolism is a response to metal exposure during germi-ation and initial plantlet elongation. This is consistent with theehaviour of roots of Lactuca sativa and Lupinus albus exposedo cadmium in hydroponic culture (Costa et al., 1997). No sig-ificant differences were observed in dry weight between treatednd control plants, but cadmium caused a decrease in relative

ater content and a reduction of 14C incorporation into amino

cids, suggesting decreased plant metabolism that may be relatedo possible Zn metabolic interference (Mckenna and Chaney,995). In this sense, other studies suggest a protective role of

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rom control and cadmium-exposed samples, not shown); (C) cotyledons fromof a 1:9 extract); (E) cotyledons from plantlets exposed to 200 mg L−1 CdCl2

(60 �L of a 1:9 extract). Cropped images.

n against the Cd response in different species (Aravind andrasad, 2005; Metwally et al., 2005).

There are few previous reports of proteomic studies of plantesponses to environmental stresses. Most used fully grownlants, hence the biological parameters evaluated as potentialarkers of pollutant exposure were often derived from leaves

n Picea abies (Davidsen, 1995), Hordeum vulgare (Tamas etl., 1997), Betula pendula (Utriainen et al., 1998). Other stud-es were performed on model plants such as O. sativa (Rakwalnd Komatsu, 2000; Rakwal et al., 1999; Hajduch et al., 2001),isum sativum (Mori, 1998), Triticum durum (Majoul et al.,000). Typically, stress proteins were detected in (H. vulgareTamas et al., 1997), Potamogeton pectinatus (Siesko et al.,997), O. sativa (Hajduch et al., 2001)), together with alterationsn redox proteins (no changes in peroxidase (Siesko et al., 1997),ut reduction and fragmentation of ribulose-1,5-bisphosphatearboxylase/oxygenase in O. sativa (Hajduch et al., 2001) andf glutathione S-transferase, involved in anthocyanin biosyn-hesis, in Zea mays (Marrs and Walbot, 1997). Studies withifferent species cell cultures demonstrated an induction of phy-ochelatins and a decrease in levels of glutathione (GSH) in theresence of Cd (Cobbett, 2000; Lee et al., 2003).

A transcriptomic study showed that several genes are acti-ated in mercuric chloride-treated Z. mays leaves, includinghose encoding glycine-rich proteins, pathogenesis-related pro-eins, chaperones and membrane proteins (Didierjean et al.,996).

This is the first proteomic analysis conduced on L. sativum,good bioindicator extensively used in environmental studies

Kazlauskiene et al., 2004; Montvydiene and Marciulioniene,004; Plaza et al., 2005). Our data suggest that, in L. sativum

E. Gianazza et al. / Environmental and Experimental Botany 59 (2007) 179–187 185

Table 1MS/MS Protein identifications of up-regulated spots in cadmium-exposed Lepidium sativum samples

Spot no. Accession no. Protein Matching tryptic peptides

2 Q9LF88 Late embryogenesis abundant protein like TTTTEPERPGLIGSVMK*

3 Q9LIF8 Jasmonate inducible protein-like TSPPFGLEGAKTNKTEYGPYGNK4 Q9ZRT9 Lea-like protein SKADETLESAKADETLESAKDK

9 Q9ZWA9 F21M11.18 protein GDVFASLAGVSQWWYNRFEAGQMEVWDHMSPELRRGDVFASLAGVSQWWYNR

10 Q9ZVY7 T25N20.16 DGQLYPEMIKLIGLEYIVTEKQCLIYDGPDANAR11 CRU3 BRANA Cruciferin cru1 precursor (11S globulin) GPFQVVRPPLRISYVVQGMGISGR13 CRU3 BRANA Cruciferin cru1 precursor (11S globulin) GPFQVVRPPLRISYVVQGMGISGR14 12S1 ARATH 12S seed storage protein precursor NPRPFYLAGNNPQGQVWLQGR15 Q9AXL9 Cruciferin subunit GPFQVVRPPLRISYVVOGMGISGR16 CRU3 BRANA Cruciferin cru1 precursor (11S globulin) ISYVVQGMGISGR17 12S1 ARATH 12S seed storage protein precursor GLYLPSFFNTAK18 LE76 BRANA Late embryogenensis abundant protein 76 TGGFLSQTGEHVK19 O22531 Low molecular weight heat-shock protein VEVEDGNILQISGER20 12S1 ARATH 12S seed storage protein precursor TNANAQINTLAGR

21 12S1 ARATH 12S seed storage protein precursor CTDNLDDPSRTNANAQINTLAGRADVYKPQLGYISTLNSYDLPILR

22 LEGB GOSHI Legumin B precursor (beta-globulin B) TNANAKISQIAGR23 12S1 ARATH 12S seed storage protein precursor TNANAQINTLAGR

24 CRU3 BRANA Cruciferin cru4 precursor (11S globulin) GQLLVVPQGFAVVKVFDQEISKGQLLVVPQGFAVVK

25 CRU3 BRANA Cruciferin BNC2 precursor (11S globulin) TNANAQINTLAGRWIEFKTNANAQINTLEAGRGLPLEVIANGYQISLEEARR

26 Q96318 12S cruciferin seed storage protein TNENAMISTLAGRIQVVNDNGQNVLDQQVQK

27 Q9AXL9 Cruciferin subunit ISYVVQGMGISGR28 Q9AXL9 Cruciferin subunit ISYVVQGMGISGR*

29 12S1 ARATH 12S seed storage protein precursor NPRPFYLAGNNPQGQVWLQGR31 12S1 ARATH 12S seed storage protein precursor DMHQKVEHIRNPRPFYLAGNNPQGQVWLQGR32 12S1 ARATH 12S seed storage protein precursor NPRPFYLAGNNPQGQVWLQGR33 12S1 ARATH 12S seed storage protein precursor NPRPFYLAGNNPQGQVWLQGR34 12S1 ARATH 12S seed storage protein precursor NPRPFYLAGNNPQGQVWLQGR35 Q9LMM4 F22L4.11 protein FSYGMAYGGGGFAISYPLAK36 12S1 ARATH 12S seed storage protein precursor SEAGRIEVWDHHAPQLR37 gi|81604 Cruciferin precursor SSQHSTQQQQAVQTNR*

38 12S1 ARATH 12S seed storage protein precursor AVYLTEGHGPASR39 12S1 ARATH 12S seed storage protein precursor SEAGRIEVWDHHAPQLR40 12S1 ARATH 12S seed storage protein precursor SEAGRIEVWDHHAPQLR

41 CRU4 BRANA Cruciferin CRU4 precursor (11S globulin) GQLLVVPQGFAVVKVFDQEISKGQLLVVPQGFAVVK

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pots excised for MS analysis are marked in Fig. 3; no matching sequence couldrimary data (high sample loads, efficient processing, reliable MS data).

lantlets, changes in protein expression are observed after expo-ure starting from low concentrations of heavy metal. This con-rms that L. sativum is a good bioindicator and proteomics is aery sensitive tool for the assessment of environment pollution.

It has been reported that general adaptation syndrome (GAS),n response to various types of environmental stress, involves thectivation of a number of mechanisms including HSPs (Leshemnd Kuiper, 1996). Our results suggest that metal stress activates

he expression of a number of proteins beside HSPs (Neumannt al., 1994; Reddy and Prasad, 1993). Future investigation wille necessary to better understand the mechanisms involved ineavy metal response.

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or NPRPFYLAGNNPQGQVWLQGR

und for 7 out of 42 spots (indicated in italics in Fig. 3), in spite of good quality

The 2-DE is hardly suitable for routine monitoring of envi-onmental pollution; however, extensive investigation and iden-ification of proteins in maps of different plant organs and atifferent growth steps will be satisfactory to identify specificiomarkers that could be easily measured by routine tests.

eferences

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